In insulin target cells, the predominantly expressed glucose transporter isoform GLUT4 recycles between distinct intracellular compartments and the plasma membrane. To characterize putative targeting signals within GLUT4 in a physiologically relevant cell type, we have analyzed the trafficking of hemagglutinin (HA)-epitope-tagged GLUT4 mutants in transiently transfected primary rat adipose cells. Mutation of the C-terminal dileucine motif (LL489/90) did not affect the cell-surface expression of HA-GLUT4. However, mutation of the N-terminal phenylalanine-based targeting sequence (F5) resulted in substantial increases, whereas deletion of 37 or 28 of the 44 C-terminal residues led to substantial decreases in cell-surface HA-GLUT4. Yeast two-hybrid analyses revealed that the N-terminal phenylalanine-based targeting signal in GLUT4 constitutes a binding site for medium chain adaptins mu1, mu2, and mu3A, implicating a role of this motif in the targeting of GLUT4 to clathrin-coated vesicles. We have used fusion of the kinase domain of Akt2 to the cytosolic C terminus of exofacially-HA-tagged GLUT4 to investigate the activity, phosphorylation state, and subcellular localization of Akt2 specifically targeted to the GLUT4-trafficking pathway in rat adipose cells. Fusion of wild-type (wt) Akt2, but not a kinase-dead (KD) mutant results in constitutive targeting of the HA-GLUT4 fusion protein to the cell surface. Insulin does not further enhance the cell-surface level of HA-GLUT4-Akt2-wt, but does stimulate the translocation of HA-GLUT4-Akt2-KD. Cell-surface HA-GLUT4-Akt2-wt is found to be phosphorylated on Ser474 and mutation of Ser474 to Ala reduces the increased basal cell-surface localization of the fusion protein. While Ser474 phosphorylation of HA-GLUT4-Akt2-KD is detected only in the insulin-stimulated state, trapping this fusion protein on the cell surface by coexpression of a dominant negative mutant dynamin does not induce Ser474 phosphorylation. Phosphorylation on Thr309 is not detectable in either HA-GLUT4-Akt2-wt or HA-GLUT4-Akt2-KD. Expression of HA-GLUT4-Akt2-wt also stimulates the translocation of cotransfected myc-GLUT4. These results demonstrate that targeting Akt2 to the GLUT4-trafficking pathway induces Akt2 activation and GLUT4 translocation. Ser474 phosphorylation is an autocatalytic reaction requiring an active kinase, and kinase activity is associated with a plasma membrane localization. Fusion of Akt2 to the C terminus of GLUT4 appears to substitute for Thr309 phosphorylation in activating the autocatalytic process. Isolation and subsequent in vitro culture of primary adipose cells are associated with down-regulation of GLUT4 mRNA and simultaneous induction of GLUT1 gene expression. Progressive loss of insulin-responsive GLUT4 contributes to the decrease in insulin-mediated glucose uptake in these cells when cultured in vitro. Here, we report that the standard procedure for isolating primary adipose cells from mouse adipose tissue triggers induction of many genes encoding inflammatory mediators including TNF-a, IL-1a, IL-6, multiple chemokines, cell adhesion molecules, acute-phase proteins, type I IL-1 receptor, and multiple transcription factors implicated in the cellular inflammatory response. Secretion of TNF-a protein was also significantly induced during the 2-h collagenase digestion of adipose tissue. However, addition of TNF-a to primary adipose cells in culture did not change the kinetics or the extent of the repression of adipose cell-abundant genes. Moreover, TNF-a-neutralizing antibody failed to block the changes in gene transcription in isolated primary adipose cells. Finally, the standard isolation procedure induced the expression of NF-kB family members and their target genes in primary adipose cells prepared from TNF-a -/- mice. Thus, these data suggest that the standard isolation procedure-triggered reprogramming of gene expression in primary adipose cells that results in decreased insulin sensitivity does not require TNF-a, but may be dependent on other inflammatory cytokines produced by these cells. We examined the role of PPAR gamma 2 and C/EBP alpha for adiponectin and aP2 gene activation in C/EBP alpha(-/-) fibroblasts by stably expressing PPAR gamma 2 or C/EBP alpha. PPAR gamma 2, but not PPAR gamma 1, mRNA markedly increased during the differentiation to adipocytes in cells expressing C/EBP alpha. Both infected cell lines differentiated to an adipocyte phenotype and the mRNA for both aP2 and adiponectin increased in parallel. However, adiponectin mRNA was considerably higher when C/EBP alpha was present, suggesting that this transcription factor is important for full gene activation. Thiazolidinediones markedly activated the gene in PPAR gamma 2-expressing cells in the absence of C/EBP alpha, suggesting that the adiponectin promoter may have functional PPAR gamma-response elements. Several observations showed that the adiponectin and aP2 genes can be differentially regulated in adipocytes: (1) Topiramate increased adiponectin mRNA levels and secretion, but did not, like the thiazolidinediones, increase aP2 expression; (2) IL-6 reduced adiponectin, but significantly increased, aP2 expression; and (3) TNFalpha inhibited adiponectin, but paradoxically increased, aP2 expression in PPAR gamma 2-infected C/EBP alpha null cells. These data show that activation of the adiponectin gene can be separated from effects on adipogenic genes.